Acetylcholine (Ach) has traditionally been described as a neurotransmitter that is widely involved in both the central and peripheral nervous systems. Over the past three decades, a variety of reports have also demonstrated that T lymphocytes secrete Ach upon stimulation, and that Ach can play a role in the regulation of T lymphocyte function. However, the precise role that Ach plays in regulating T cell function has not been thoroughly investigated and little is known concerning the role of Ach in regulation of CD8+ T cells. The preliminary data presented in this grant demonstrate that depletion of Ach prevents the differentiation of naive CD8+ T cells into cytolytic T lymphocytes (CTL) in vitro. In addition, CD8+ T cells from mice with a targeted deletion the M1 Ach receptor likewise fail to differentiate into CTL in vitro. Based upon these novel findings, we hypothesize that Ach represents a previously unappreciated autocrine and/or paracrine requirement for CTL generation that exerts its effects through the MI Ach receptor. The focus of this grant is an in depth testing of this hypothesis both in vitro and in vivo using Ach depletion and the M1 knockout mouse. Two specific aims are presented: i. Characterize the Defects in CD8+ T cells from M1 KO mice in vitro. ii. Assess CD8+ T cell responses of M1 KO mice in vivo. Since CTL are central to the eradication of infection by numerous pathogens, involved in immune based elimination of neo-plastic tissues, and can be responsible for significant pathology in both transplant rejection and autoimmune disorders, understanding the role of Ach in CTL development has broad applicability to numerous human diseases. The proposed studies will rigorously test our hypothesis and evaluate the M1 knockout mouse as an in vivo model for studying the effects of Ach on CTL development. These investigations will assess the importance of Ach in regulating CD8+ T cell function and lay the groundwork for subsequent development of a full scale in depth research program aimed at elucidating the effects and mechanisms by which Ach regulates T cell based immunology.